MIP-1 alpha and TGF-beta production in CD34+progenitor-stromal cell coculture systems: Effects of progenitor isolation method and cell-cell contact

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a C-C chemokine wh ich has antiproliferative effects on early hematopoietic progenitors and st imulatory effects on later progenitors. It also possesses chemotactic and a ctivating properties for monocytes, macrophages, and T-cells. CD34+ progeni tors isolated utilizing an avidin-biotin immunoadsorption column produced s ignificant amounts of MIP-1 alpha from 24 h onward when cultured in medium with 10% fetal calf serum (>200 pg/ml). Such production persisted through 9 6 h of culture and was greater when such progenitors were cocultured with a preformed marrow stromal layer (4000 pg/ml at 24 h). The production of MIP -1 alpha declined over time of coculture with stromal layers, and stromal l ayers themselves produced minimal MIP-1 alpha as detected by ELISA: <100 pg /ml. In contrast, CD34+ cells isolated by flow cytometry or by magnetic bea d adsorption produced minimal MIP-1<alpha> (0-30 pg/ml). MIP-1 alpha produc tion also increased when cells isolated by these two methods were coculture d with stromal layers. The difference in MIP-1 alpha production could not b e accounted for by differences in purity of the CD34+ population between is olation methods nor on the basis of monocytic or lymphocytic contamination as assessed by the presence of CD 14 or CD3 positive cells. CD34+ cells iso lated by immune adsorption had increased expression of endothelial and mese nchymal associated antigens, however, suggesting that this subpopulation mi ght account for the MIP-1 alpha production observed. Freshly isolated CD34 cells expressed MIP-1 alpha message as assessed by RT-PCR and by in situ h ybridization. Coculture of CD34+ cells isolated by any means with stromal c ells increased transforming growth factor-beta (TGF-beta) production, in th is case by the stromal layer itself. Both MIP-1 alpha and TGF-beta have bee n found to influence cell cycle status and proliferation status of early he matopoietic progenitors, and both have potential effects on accessory cell function. These studies indicate that progenitor-stromal cell interactions may influence local cytokine output, thus potentially influencing progenito r cycling status and accessory cell activation. The method of isolation of CD34+ progenitors may influence secretion of certain cytokines and chemokin es. (C) 2000 Academic Press.